Abp pab 10683

Tumor samples were fixed with 4% paraformaldehyde, processed and embedded in paraffin. Standard immunohistochemical techniques were used according to the manufacturer’s recommendations (Vector Laboratories) using antibodies against Notch3 (Allele biotechnology, ABP-PAB-10683), β-catenin (Cell Signaling Technology, 2677), avidin DH: biotinylated horseradish peroxidase H complex with 3,3′-diaminobenzidine (Polysciences), and Mayer’s hematoxylin (Fisher Scientific). For IF staining, Notch3 primary antibodies and Alexa 488/568 secondary antibodies (Life Technologies, NY) were used for labeling. Xenograft tumor sections were de-waxed by heating on a slide warmer at 60 °C for 15 min.

HCC827 and HCC4006 cells were seeded in eight-well chamber slides and treated with vehicle, DMSO or erlotinib for 6 days. Cells were fixed with 4% paraformaldehyde and incubated with primary antibodies against Notch3 rabbit polyclonal antibody (Allele biotechnology, ABP-PAB-10683), β-catenin (L54E2) Mouse mAb-Alexa Fluor 555 conjugate (Cell Signaling Technology, 2677) alone or together overnight at 4 °C. After washing, cells were stained with Alexa Fluor- 488 or 594 conjugated secondary IgG antibodies. To detect the nuclei, the slides were mounted with Vectashield anti-fade Mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). Cells were visualized using an Olympus FV1000 Filter confocal microscope fitted with a ×40 oil objective.